GB2157430A

GB2157430A - A method for the fluorometric determination of catecholamines

Info

Publication number: GB2157430A
Application number: GB08508287A
Authority: GB
Inventors: Yosuke Ohkura
Original assignee: Toyo Soda Manufacturing Co Ltd
Current assignee: Tosoh Corp
Priority date: 1984-03-29
Filing date: 1985-03-29
Publication date: 1985-10-23
Also published as: JPS60205262A; DE3511356A1; US4705757A; GB2157430B; DE3511356C2; JPH046897B2; GB8508287D0

Description

1 GB 2 157 430A 1

SPECIFICATION

A method for the fluorometric analysis of catecholamines The present invention relates to a method for the fluorometric analysis of catecholamine using 5 1,2-diphenylethyiene diamine.

Catecholamine is a general term for biogenic amines having a 3,4dihydroxyphenyl group. In particular, catecholamines such as norepinephrine, epinephrine, dopamine and the like which are metabolites of tyrosine perform an important function as the adrenal medulla hormones or as the neurotransmittors at the end of sympathetic nerves. Therefore, the analysis of catecholamine 10 is indispensable for the studies of endocrinium and the nervous system. However, the content of catecholamine is extremely low in the tisue; it is not more than 1 / 104 of amino acid, so an analytical method with high sensitivity is required. Recently, high sensitivity fluorometric analysis of catecholamine has attracted attention for clinical application.

Although a method is known for measuring the native fluorescence of catecholamine at pH 1, 15 it is not specific for catecholamine and its sensitivity is not so high. One method which is adopted most popularly is to determine the fluorescence of trihydroxyindole (THI) formed by treating catecholamine with an oxidizing agent. This method has a high sensitivity particularly to norepinephrine and epinephrine. But, dopamine, resistant to oxidation cannot be determined with a sufficient sensitivity even by the THI method. Another method is also known which determines catecholamine through a condensation reaction with ethylenedia mine, but it has shortcomings in that the procedure is complicated and its sensitivity is low.

As a result of diligent investigations aiming at the development of a high sensitive fluorescence deriving reagent for biological catecholamine, the present inventors have surprisin gly found that 1,2-diphenylethylenediamine (hereinafter abbreviated to DPE) reacts with 25 catecholamine to give a strong fluoressing compound.

Thus, the invention provides a method for the fluorometric analysis of catecholamines using DPE as a fluorescence-deriving reagent, wherein, after the addition of catecholamine into a solution of DPE containing an oxidizing agent at pH 4 to 10, the mixture is allowed to stand for more than 1 minute at a temperature of higher than 1 5C to make catecholamine fluorescent. 30 The synthesis of DPE of the invention is conducted as follows: To N- benzyi-N'-benzyiidene meso. 1, 2-diphenylethylene-dia mine obtained by refluxing ammonium acetate and benzaldenyde for several hours are added water and concentrated sulfuric acid. After allowing the mixture to react by heating and by passing steam for several hours, the mixture is neutralised and cooled with ice. Thereby, crystals of DPE can easily be obtained (J. Inorg. Nucl. Chem 27, (1965) 270-271).

The concentration of DPE in the reaction is higher than 1 OmM, preferably 0. 1 to 0.3 M. A concentration below- 1 OmM is not preferable because of a rapid decrease in reaction rate.

Practically, a concentration of 0. 1 to 0. 3 M is preferable where high reaction rate is obtainable.

Since a solution of DPE is a strong base, the pH value of the solution of DPE containing the 40 oxidizing reagent is adjusted to pH 4 to 10, preferably to pH 5 to 9 with acid according to the invention.

As the acids, inorganic acid such as hydrochloric acid and nitric acid, and organic acids such as formic acid and acetic acid can be mentioned, but hydrochloric acid and acetic acid are particularly preferable. Moreover, Bicine and Britton-Robinson buffer solution, for example may 45 be used to keep the pH value constant.

At a pH below 4, the reaction does not take place or the reaction rate is low, and, in at pH above 10, the reaction velocity decreases. In practice, a range of pH 5 to 9 is preferable where the reaction velocity takes maximum value.

As suitable oxidizing reagents, potassium ferricyanide, sodium hypochlorite, hydrogen perox- 50 ide, and oxygen, for example can be mentioned, but potassium ferricyanide is particularly preferable. The concentration of the oxidizing reagent should be higher than 0.5 mM, preferably 1.8 to 3.0 mM. A concentration of oxidizing reagent below 0.5 mM is not preferable since the reaction rate is low and the reaction does not take place adequately. Moreover, in a concentration range over 0.5 mM, the reaction rate decreases gradually with an increase in the 55 concentration, but a range of 1.8 to 3.0 mM is preferable where the maximum and constant value is obtainable.

The temperature of the reaction should be higher than 15 C, preferably 30 to 1 OWC, and the period of time for allowing to stand is more than 5 minutes, preferably 10 to 40 minutes. At a temperature of not higher than 1 WC, the reaction rate is low and it takes a long time to attain 60 the maximum value, and, with a standing time of not more than 5 minutes, the reaction does not progress sufficiently.

The fluorescent body obtained through the reaction mentioned above is stable for 2 hours at least.

Moreover, in the aforementioned reaction, in the presence of a catalyst in DPE solution 65 2 GB 2 157 430A 2 containing the oxidizing reagent, the reaction proceeds much faster. As oxidation promoters, ordinary oxidation promoters can be used without any trouble. Among them, glycine, ethanol, acetonitrile and acetone are preferable, but glycine is particularly preferable. 5 The amount of the oxidation promoter to be coexistent is preferably more than 0.05 M, most 5 preferably 0.2 to 0.8 M. If the oxidation promoter is used in an amount less than 0.05 M then the reaction rate is low and the reaction does not take place sufficiently. Practically, a range of 0.2 to 0.8M is preferable where the reaction rate is high. As described above, according to the invention, the fluorescence-taged substance of the catecholamines can be obtained easily using DPE as fluorescence deriving. Therefore, this is applicable not only to the manual method, but also to the precolumn or postcolumn deriving method in the high performance liquid chromatography, and can be said to be an excellent method of wide application for fluorescent analysis.

The invention is illustrated with reference to the following examples.

Example 1

The manual fluorometric determination was investigated using norepinephrine, epinephrine, dopamine and isoproternol. DPE was synthesized by the method shown below.

To 1 Og of N-benzoyl-N-benzyiidine-meso- 1, 2-d iphenylethylened ia mine obtained in a yield of 44% by refluxing for 3 hours to allow 40 9 of ammonium acetate to react with 100 mi of benzaldehyde were added 100 mI of water and 54 mi of concentrated suifuric acid. The resulting mixture was allowed to react for 4 hours by heating and introducing steam. After completion of the reaction, the mixture was neutralized with ammonia and cooled with ice to obtain the crystals of 1,2-diphenylethylenediamine.

To a solution of DPE prepared by adding 20 m] of 0.05 M Bicine buffer solution (pH 7.0) 25 containing 0.5 M glycine, 0.1 mi of 0.1 M DPE solution (ethanol solvent) and 0.1 mi of 2.5 mM potassium ferricyanide solution in sequence were added various types of catecholamines.

After standing for 30 minutes at 37'C, the fluorescence intensity was measured. The optimal wavelength of the fluorescence defection is shown in Table 1. The lower limit of the determination (S/ N = 2) was 2 X 19 11 M for all the catecholamines. Moreover, when simulta- 30 neous measurements were made twenty times using 1 X 10-6 M solution, the coefficient of variation was found to be 1 % or so for all catecholamines. DPE reacted selectively only with the compounds having a catechol skeleton as shown in Table 1, and there were no significant differences in the intensity of fluorescence among norepinephrine, epinephrine, dopamine and isoproterenol. When compared with the THI method in which the fluorescence of trihydroxy- 35 indole formed by treating catecholamine with oxidizing agent is measured, in regard to norepinephrine, epinephrine and isoproterenol, about 10 times and, in regard to dopamine, about 500 times as strong fluorescences were observed as those by THI method. Thus the method of the invention has a high sensitivity. No DPE reacted with the biological constituents such as sugars, keto acids, amino acids, nucleic acid bases, steroids, polyaminocarboxylic acids, 40 alcohols and aldehydes, and it was confirmed to be a selective reagent for catecholamines.

GB 2 157 430A Table 1 Fluorescence characteristic and limit of detection of catechol compounds reacted with DPE Catechol compound Excitation Fluorescence Relative Limit of wavelength wavelength intensity detection ex (nm);k em (nin) of f luor- pmol/ml escence Catechol 345 480 14 100 Pyrogallol 330 475 3 500 3,4-Dihydroxybenzyl- 340 470 32 so amine 3,4-Dihydroxy- 330 480 3 500 benzoic acid Norephinephrine 340 480 100 is Epinephrine 350 496 64 20 Dopamine 347 470 84 20 Isoproterenol 356 497 104 10 Dopa 348 480 19 80 3,4-Dihydroxyphenyl 345 477 72 20 acetic acid 3,4-Dihydroxyman- 350 470 9 150 delic acid 3,4-Dihydroxyphenyl- 347 478 95 15 ethylene glycol 2-Hydroxyestrone 350 475 6 300 4-Hydroxyestrone 350 475 1 1800 3 Example 2

The manual determination of the fluorescence was investigated using norephinephrine, epinephrine, dopamine and isoproterenol.

To 20 mi of solution made by adding acetic acid to 0.005 M 1,2diphenylethylenediamine 45 involving the sample for adjusting pH to 7.0 were added 2.5 mM of potassium ferricyanide.

After allowing the mixture to stand for 30 minutes at 70C, the fluorescence was measured. The optimal wavelength for the fluorescence measurement was shown in Table 1. The limit of the detection (S/N = 2) was 8 X 10-8 M for all of cetecholamines. Moreover, when simultaneous measurements were made twenty times using 1 X 10-6 M solution, the coefficient of variation 50 was found to be 1 % or so for all of catecholamines.

Example 3

When many constituents are present in the biological samples etc., the high-performance liquid chromatography (HPLC) is powerful as a method for the analysis. Therefore, the analysis 55 of catecholamines was carried out by the precolumn deriving method used DPE.

To the sample solution (1.0 mi) were added 2.0 mi of 0.05 M Bicine buffer solution (pH 7.0) containing 0.5 M glycine, 0.1 mi of 0.1 M DPE solution (ethanol solvent) and 0.1 mi of 0.08 % (W/V) potassium ferricyanide solution in sequence. After allowing to stand for more than 30 minutes at 37'C, 100 td of the reaction liquid were injected into HPLC apparatus. Conditions of 60 HPLC were as follows: column; Ultrasphere-ODS (4.60X 150 mm, Beckman), eluate; CH3CN-H,0 (47.5: 52.5 W/V), flow rate 1 mi/min. For the fluorescence detection, Shimazu FLD-1 (excitation 300-400 nm, fluorescence 450-800 nm) was employed. Under these conditions, reversed-phase partition type column as mentioned above, norepinephrine, epine- phrine and isoproterenol gave a single peak respectively. Although dopamine gave two peaks of 65 4 GB2157430A 4 an intesity ratio of 9: 1, the determination was possible with either of them. The elution was completed within 12 minutes, and the lower limit of detection was 10 pg at SN ratio of 2.

Example 4

The precolumn deriving method with a reaction prior to the injection into H PLC as in Example 5 3 necessitates a complicated procedure and is troublesome because of the manual practice of the reaction. Therefore, more powerful analysis of catecholamine was carried out which is automatable by the postcolumn deriving method allowing to develop after the separation with HPLC.

Into the column eluate, containing (1) (TSK-gel Ether 250, Toyo Soda Kogyo Co., Ltd) mM of 10 sodium acetate, 30 mM of sodium perchlorate and 5% ethanol was injected the biological sample. After allowed catecholamine to absorb to the column (1), this was washed with purified water. Then, the catecholamine constituents were transferred to the column (2) (TSK-gel SP 2SW, Toyo Soda Kogyo Co., Ltd) with aqueous solution of 20 mM sodium acetate /aceto n i trile = 50/50. Following this, catecholamine was separated into three constituents in the 1 column (3) (TSK-gel Catecholpak, Toyo Soda Kogyo Co., Ltd) using aqueous solution of 0.2 M sodium chloride containing 10% acetonitrile as an eluate for H PLC. These three separated constituents were mixed with aqueous solution of 0.01 M DPE/ethanol = 50/50 and aqueous solution of 0.5 Mm potassium ferricyanide/ethanol = 50/50, and made up the fluorescence emitting constituents in the reaction coil. These fluorescence-emitting constituents were detected 20 by the use of fluorescence detector (FS-8000, Toyo Soda Kogyo Co., Ltd).

Figure 1 shows the chromatograms of the standard sample (each 100 pg/mi) and the serum sample measured. The limit of the detection is 2 pg at SN ratio of 2.

4. Brief Description of the Drawing

Figure la and b are chromatograms of serum and a standard sample measured according to 25 the invention respectively.

1. Epinephrine 2. Norepinephrine 3. Dopamine

Claims

CLAIMS 1. A method for the fluorometric analysis of catecholamines wherein

after the addition of catecholamine to a solution of 1,2diphenylethylenediamine containing an oxidizing agent at pH 4 to 10, the mixture is allowed to stand for more than 1 minute at a temperature above 1 WC to make catecholamine fluorescent.
2. A method for the fluoreometric analysis of catecholamines according to claim 1, wherein the solution of 1,2-diphenylethylenediamine contains an oxidation promoter.
3. A method for the fluorometric analysis of catecholamines according to claim 1 or 2, wherein the concentration of 1,2-diphenylethylenediamine is from 0.1 to 0. 3M.
4. A method for the fluorometric analysis of catecholamines according to any of claims 1 to 40 3, wherein the pH is from 5 to 9.
5. A method for the fluorometric analysis of catecholamines according to any of claims 1 to 4, wherein the pH is adjusted by addition of hydrochloric or acetic acid.
6. A method for the fluorometric analysis of catecholamines according to any of claims 1 to 5, wherein the concentration of the oxidizing agent is 1.8 to 3.0 mM.
7. A method for the fluorometric analysis of catecholamines according to any of claims 1 to 6, wherein an oxidation promoter is employed.
8. A method for the fluorometric analysis of catecholamines according to claim 7, wherein the oxidation promoter is glycine, ethanol, acetonitrile or acetone.
9. A method for the fluorometric analysis of catecholamines according to claim 1 substan- 50 tially as herein described with reference to the specific Examples.

Printed in the United Kingdom for Her Majesty's Stationery Office, Dd 8818935. 1985, 4235. Published at The Patent Office. 25 Southampton Buildings. London, WC2A l AY, from which copies may be obtained.